Hydrophobic interaction membrane chromatography for plasmid DNA purification: Design and optimization

Chromatography is one of the key operations in the downstream processing of plasmid DNA (pDNA). However, the increased demand for highly purified pDNA experienced in recent years has made clear the need for alternative processes capable of retaining the advantages of conventional chromatography, such as selectivity, while providing increased throughput at a lower cost. The work presented in this article outlines the development and optimization of an alternative hydrophobic interaction membrane chromatography process for the purification of pDNA. The studies included the modification of functionalized membrane supports with a linear alkyl chain ligand and the testing of chromatographic performance of these membranes. Three modification procedures were tested and the membranes were screened for their capacity and selectivity. The modified membranes could separate the model plasmid pVAX1‐LacZ (6050 bp) from impurities in clarified Escherichia coli cell lysates (specifically RNA), with good resolution. Subsequent optimization of elution profiles with the best‐performing modified membrane, resulted in a high purification factor of 4.7, competitive with its bead process counterpart, and a plasmid yield of 73%.     

Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry

A lipidomics strategy, combining high resolution reversed-phase liquid chromatography (RPLC) with high resolution quadrupole time-of-flight mass spectrometry (QqTOF), is described. The method has carefully been assessed in both a qualitative and a quantitative fashion utilizing human blood plasma. The inherent low technical variability associated with the lipidomics method allows to measure 65% of the features with an intensity RSD value below 10%. Blood plasma lipid spike-in experiments demonstrate that relative concentration differences smaller than 25% can readily be revealed by means of a t-test. Utilizing an advanced identification strategy, it is shown that the detected features mainly originate from (lyso-)phospholipids, sphingolipids, mono-, di- and triacylglycerols and cholesterol esters. The high resolution offered by the up-front RPLC step further allows to discriminate various isomeric species associated with the different lipid classes. The added value of utilizing a Jetstream electrospray ionization (ESI) source over a regular ESI source in lipidomics is for the first time demonstrated. In addition, the application of ultra high performance LC (UHPLC) up to 1200 bar to extend the peak capacity or increase productivity is discussed.     

A Validated LC Method for the Quantitation of Cefotaxime in pH-Sensitive Nanoparticles

A sensitive and rapid routine LC method was validated for measuring cefotaxime incorporated in three different pH-sensitive nanoparticles. The drug was chromatographed on a C18 reversed-phase column; the mobile phase used was 0.05 M aqueous ammonium acetate, acetonitrile and tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5 with acetic acid. The flow rate was 1 mL min^?1 and cefotaxime was quantified at 254 nm, with a sensitivity range of 0.005 AUFS. The validated method was specific, linear (R ² ≥ 0.999), precise and accurate in a concentration range of 0.2–50.0 μg mL^?1. The method was rapid, selective and suitable for evaluation of cefotaxime in pH-sensitive Eudragit nanoparticles.     

Evaluation of a new polymeric stationary phase with reversed-phase properties for high temperature liquid chromatography

The performance of a polymeric stationary phase with reversed-phase properties (ET-RP1) was evaluated for LC separations at elevated temperature. The most significant observation was that the reduced plate height (h) decreased from 3.4 at 25 °C (optimal flow 0.5 mL/min) to 2.4 at 150 °C (optimal flow 2.5 mL/min) which is comparable to the efficiency obtained with silica-based reversed-phase columns of 4.6 mm ID operated at 0.8 mL/min. The phase showed no deterioration after long use at 150 °C within the pH range 1–9. Catalytic activity originating from the stationary phase material, e.g. as experienced on zirconium columns operated at elevated temperature, was absent. The performance of ET-RP1 is illustrated with the analysis of some pharmaceutical samples by LC and LC–MS. Operation at elevated temperature also allows to reduce the amount of organic modifier or to replace acetonitrile and methanol by the biodegradable ethanol.     

TLD measurements and Monte Carlo simulations for glandular dose and scatter fraction assessment in mammography: A comparative study

The main purpose of this study was to validate and compare Mean Glandular Dose (MGD) values obtained using Monte Carlo simulations with experimental values obtained from Entrance Surface Dose (ESD) and depth dose measurements performed in a Hospital mammography unit. ESD and depth dose were measured using ThermoLuminescent Dosimeters (TLDs), and a tissue equivalent mammography phantom recommended by the American College of Radiology (ACR). Measurements and Monte Carlo simulations were also compared with the MGD calculated using the Automatic Exposure Control (AEC) system of the mammographic unit. In the simulations the Doppler energy broadening effect was also taken into account. The simulated ESD are about 5%–10% higher than the measured ESD values. The deviation between the measured and simulated MGD values in the phantom is of about 15%. The MGD evaluated using the AEC system is smaller both with respect to the Monte Carlo simulation and experimental result by a factor of about 15% and 25% respectively. Moreover the BackScatter Factor (BSF) estimated by Monte Carlo simulations was used for the MGD calculation according to the Wu’s method. Finally the inclusion of the energy broadening effect on MGD calculation produces negligible variations on the simulated results.     

Flowing liquid crystal simulating the Schwarzschild metric

We show how to simulate the equatorial section of the Schwarzschild metric through a flowing liquid crystal in its nematic phase. Inside a liquid crystal in the nematic phase, a traveling light ray feels an effective metric, whose properties are linked to perpendicular and parallel refractive indexes, n _o and n _e respectively, of the rod-like molecule of the liquid crystal. As these indexes depend on the scalar order parameter of the liquid crystal, the Beris-Edwards hydrodynamic theory is used to connect the order parameter with the velocity of a liquid crystal flow at each point. This way we calculate a radial velocity profile that simulates the equatorial section of the Schwarzschild metric, in the region outside of Schwarzschild’s radius, in the nematic phase of the liquid crystal. In our model, the higher flow velocity can be on the order of some meters per second.     

Modification of the electrical properties in Fe-Al2O3 granular thin films due to increased bias potential

We studied the electrical properties in Fe-Al₂O₃ granular films when the injected direct current or bias potential are varied in the low-field regime (eΔV?k_BT). Measurements of the electrical resistance as a function of temperature and applied bias at different temperatures were performed. We found that the electrical properties are best described using variable range hopping. The variation in resistance showed unexpected characteristics in distinct regions of potential. These regions of potential could be due to modification of the electronic localization length. We have shown that the electrical resistance decreases when the applied bias and/or current increases. We associate this behavior of the resistance with the activation of new electronic paths. The total resistance of our samples is reduced as additional parallel electronic paths are formed.     

3D elemental distribution images in biological samples by XRFµCT

An X‐ray transmission microtomography (CT) system combined with an X‐ray fluorescence microtomography (XRFµCT) system was implemented in the Brazilian Synchrotron Light Laboratory (LNLS), Campinas, Brazil. The aim of this work was to determine the elemental distribution in biological samples (breast, prostate and lung samples) in order to verify the concentration of some elements correlated with characteristics and pathology of each tissue observed by the transmission CT. The experiments were performed at the X‐ray fluorescence beamline (D09B‐XRF) of the Brazilian Synchrotron Light Laboratory, Campinas, Brazil. A quasi‐monochromatic beam produced by a multilayer monochromator was used as an incident beam. The sample was placed on a high‐precision goniometer and translation stages that allow rotating as well as translating it perpendicularly to the beam. The fluorescence photons were collected with an energy dispersive HPGe detector placed at 90° to the incident beam, while transmitted photons were detected with a fast Na(Tl) scintillation counter placed behind the sample on the beam path. The CT images were reconstructed using a filtered‐back projection algorithm and the XRFµCT images were reconstructed using a filtered‐back projection algorithm with absorption corrections. The 3D images were reconstructed using the 3D‐DOCTOR software. Results from the 3D visualization showed that the distribution of iron, copper and zinc is different and heterogeneous from the analyzed samples. Copyright © 2011 John Wiley & Sons, Ltd.     

Comparison of Digital Imaging Systems for Neutron Radiography

The characteristics of three digital imaging systems for neutron radiography purposes have been compared. Two of them make use of films, CR-39 and Kodak AA, and the third makes use of a LiF scintillator, for image registration. The irradiations were performed in the neutron radiography facility installed at the IEA-R1 nuclear research reactor of IPEN-CNEN/SP. According to the obtained results, the system based on CR-39 is the slowest to obtain an image, and the best in terms of resolution but the worse in terms of contrast. The system based on Kodak AA is faster than the prior, exhibits good resolution and contrast. The system based on the scintillator is the fastest to obtain an image, and best in terms of contrast but the worse in terms of resolution.     

Strange stars properties calculated in the framework of the Field Correlator Method

We calculate the strange star properties in the framework of the Field Correlator Method. We find that for gluon condensate values G₂$G_2$ in the range 0.006–0.007 GeV⁴$0.006\text{–}0.007{\text{GeV}}^4$, which give a critical temperature Tc∼170 MeV$T_c\sim 170\text{MeV}$ at μc=0${\mu }_c=0$, the sequences of strange stars are compatible with some of the semi-empirical mass–radius relations and data obtained from astrophysical observations.